RUI (MCB - Genetic Mechanisms): Molecular analysis of two interacting components of the conjugation machinery of Bacillus subtilis

RUI（MCB - 遗传机制）：枯草芽孢杆菌接合机制的两个相互作用成分的分子分析

• 批准号: 1613920
• 负责人: Melanie Berkmen
• 金额: $ 36.12万
• 依托单位: Suffolk University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1613920&HistoricalAwards=false
• 关键词: RUI; MCB; Genetic; Mechanisms; Molecular

Bacteria have the remarkable ability to acquire new genes in a process known as conjugation, which plays a profound role in bacterial evolution and adaptation to different environments. During conjugation, DNA is transferred from one cell to another through a specialized translocation channel. Many of the molecular mechanisms behind the conjugation process remain a mystery. This project will characterize two critical interacting protein components of the conjugation machinery of the bacterium Bacillus subtilis, providing insight into how they function, interact, and localize. The research will be designed and conducted by 12 highly qualified and diverse undergraduate students. By attaining this independent, inquiry-driven research experience, students are more likely to remain in science and gain employment or entrance to top-tier graduate programs after graduation. This project will also improve science education by enabling a professor from a diverse community college to do research and learn new techniques.The subject of this study is a specialized DNA translocation channel known as a Type IV secretion system (T4SS). While the T4SSs found in several Gram-negative bacteria have been well-studied, much less is known about those of Gram-positive bacteria. This project aims to examine the conjugation machinery of Gram-positive bacteria using the integrative and conjugative element ICEBs1 of B. subtilis as a model system. Prior results indicate that the ICEBs1 T4SS is composed of ConE and ConB, among other proteins. ConE is a peripheral membrane ATPase that likely energizes DNA transfer and/or channel assembly. The bitopic membrane protein ConB interacts with ConE and is required for its association with the membrane. ConB and ConE will be characterized by a variety of methods including mutagenesis, conjugation assays, fluorescence microscopy, and biochemistry. This research will define the conserved sequence motifs and domains of ConB and ConE that are required for conjugation, interaction, enzymatic function, and oligomerization. As these proteins are conserved, the findings will apply to the conjugation machinery of numerous Gram-positive bacteria and provide a deeper understanding of a major mechanism mediating horizontal gene transfer.

细菌具有在称为接合的过程中获得新基因的非凡能力，这在细菌进化和适应不同环境中起着深远的作用。在接合过程中，DNA通过专门的易位通道从一个细胞转移到另一个细胞。共轭过程背后的许多分子机制仍然是一个谜。该项目将表征细菌枯草芽孢杆菌的接合机制的两个关键相互作用的蛋白质组分，从而深入了解它们如何发挥功能，相互作用和定位。该研究将由12名高素质和多样化的本科生设计和进行。通过获得这种独立的，探究驱动的研究经验，学生更有可能留在科学和毕业后获得就业或进入顶级研究生课程。该项目还将通过让来自多元化社区大学的教授进行研究和学习新技术来改善科学教育。本研究的主题是一种被称为IV型分泌系统（T4 SS）的特殊DNA易位通道。虽然在几种革兰氏阴性细菌中发现的T4 SS已经得到了很好的研究，但对革兰氏阳性细菌的T4 SS知之甚少。本项目旨在利用B的整合接合元件ICEBs 1研究革兰氏阳性菌的接合机制。subtilis作为模型系统。之前的结果表明，ICEB 1 T4 SS由ConE和ConB等蛋白质组成。ConE是一种外周膜ATP酶，可能为DNA转移和/或通道组装提供能量。双位膜蛋白ConB与ConE相互作用，并且是其与膜结合所必需的。ConB和ConE将通过多种方法进行表征，包括诱变、缀合测定、荧光显微镜和生物化学。本研究将确定ConB和ConE的保守序列基序和结构域，这些基序和结构域是缀合、相互作用、酶功能和寡聚化所必需的。由于这些蛋白质是保守的，这些发现将适用于许多革兰氏阳性细菌的接合机制，并提供了介导水平基因转移的主要机制的更深入的了解。